Changes in bacterial community composition in the uterus of Holstein cow with endometritis before and after treatment with oxytetracycline

It is important to study the bacteria that cause endometritis to identify effective therapeutic drugs for dairy cows. In this study, 20% oxytetracycline was used to treat Holstein cows (n = 6) with severe endometritis. Additional 10 Holstein cows (5 for healthy cows, 5 for cows with mild endometritis) were also selected. At the same time, changes in bacterial communities were monitored by high-throughput sequencing. The results show that Escherichia coli, Staphylococcus aureus and other common pathogenic bacteria could be detected by traditional methods in cows both with and without endometritis. However, 16S sequencing results show that changes in the abundance of these bacteria were not significant. Endometritis is often caused by mixed infections in the uterus. Oxytetracycline did not completely remove existing bacteria. However, oxytetracycline could effectively inhibit endometritis and had a significant inhibitory effect on the genera Bacteroides, Trueperella, Peptoniphilus, Parvimonas, Porphyromonas, and Fusobacterium but had no significant inhibitory effect on the bacterial genera Marinospirillum, Erysipelothrix, and Enteractinococcus. During oxytetracycline treatment, the cell motility, endocrine system, exogenous system, glycan biosynthesis and metabolism, lipid metabolism, metabolism of terpenoids, polyketides, cofactors and vitamins, signal transduction, and transport and catabolism pathways were affected.

Oxytetracycline (OTC) has good efficacy as an antibiotic for the treatment of endometritis [14][15][16] .OTC is an inexpensive, broad-spectrum antibiotic that is active against a wide variety of bacteria.OTC binds to the 30S ribosomal subunit and prevents the formation of an aminoacyl-tRNA-ribosome complex and further interferes with the ability of bacteria to produce proteins that are essential for bacteria to grow and multiply.However, some strains of bacteria have developed resistance to OTC, which has reduced its effectiveness for treating some types of infections [17][18][19] .Therefore, to effectively utilize the efficacy of OTC and treat endometritis, it is important to explore the bacterial communities that cause infection and are sensitive to OTC.
With the development of biotechnology, high-throughput sequencing has been widely used in clinical veterinary fields due to its high throughput, short sequencing time and low cost.When faced with certain clinical statuses, such as increased types of diseases, accelerated pathogen mutations, secondary infections, mixed infections, and latent infections, high-throughput sequencing has shown advantages in the areas of disease surveillance, rapid and accurate diagnosis, pathological research, and drug screening [20][21][22] .
This study used high-throughput sequencing to analyze changes in the bacterial community composition of bovine uterus contents in healthy Holstein cows, cows with endometritis, and cows with endometritis treated by OTC.Bacteria at the genus level that are easy or difficult to inhibit with OTC and the pathways that OTC affects were evaluated.These findings provide a theoretical basis for analyzing the pathogenesis of endometritis and the rational use of OTC.

Pathogen identification in cultured bacteria
The detection rate of corresponding bacteria with common culture techniques in all four groups is shown in Table 1.Briefly, the bacteria detection rates (the number of positive samples to the number of all samples) between the NT and NC groups were similar.The detection rates of Escherichia coli, Staphylococcus aureus, Staphylococcus haemolyticus, and Streptococcus uberis were greater than 83%, while the detection rates of other bacteria were no less than 33%.The severity of endometritis can be quickly distinguished through visual observation and ultrasound methods (Fig. 1A-J).Microscopic examination results also showed that the cultures in this study had mixed infections of Escherichia coli, Staphylococcus sp., and Streptococcus sp. (Fig. 1K, L).In individuals with severe endometritis (BT group), the detection rate of all mentioned bacteria in Table 1 is relatively higher.After treatment with oxytetracycline (AT group), the detection rate of mentioned bacteria was reduced.However, the related bacteria could still be detected in all samples.

Alpha-diversity of bovine uterine bacterial communities
The sequencing reads of the bacterial 16S rRNA gene resulted in 1,488,649 clean tags from 22 samples.The evaluation results of each sample's sequencing data are shown in Supplementary Table 1.These high-quality sequences clustered into an average of 1,117 bacterial OTUs.Numbers of identified phyla, classes, orders, families, genera, and species from these OTUs in all samples are shown in Supplementary Table 2.

Bacterial beta-diversity
A Bray-Curtis-based NMDS plot of all samples revealed a separation among the four groups (Fig. 3C).The Bray-Curtis-based distance and abundance metrics showed that samples belonging to the BT group had an obviously different distribution compared with that of the other three groups.A UPGMA-based hierarchical clustering analysis confirmed the alpha-diversity analysis results, which showed that each group had unique communities compared with those of the other groups among the four groups (Fig. 4).

Changes in the bacterial community in the uterus before and after treatment with OTC
The results of LEfSe analysis indicated that the bacterial communities with significant abundance changes in the four groups included 6 phyla, 10 classes, 15 orders, 24 families, 28 genera, and 1 species (Fig. 5 and Supplementary Table 3).Metagenomic analysis showed that the bacterial composition varied in uteruses with endometritis after OTC treatment.We first clarified the changes in the bacterial communities at the phylum level (Fig. 6A-C).The relative abundance of Fusobacteria, Firmicutes, Bacteroidetes, Spirochaetae, Proteobacteria, Saccharibacteria, Chloroflexi,

Changes in functional pathways of bacterial communities among the different groups
As shown in Fig. 6D, there were 11 biological pathways (including excretory system, signal transduction, cell motility, digestive system, metabolism of terpenoids and polyketides, endocrine system, glycan biosynthesis and metabolism, lipid metabolism, metabolism of cofactors and vitamins, cancers: overview, and transport and catabolism) involved in bacterial communities with significant abundance changes between the BT and AT groups.

Discussion
OTC has a good therapeutic effect on endometritis, but some bacteria are resistant to OTC.However, most research on the use of oxytetracycline (OTC) in the treatment of endometritis focus on specific flora, especially the culturable bacteria 23,24 .The inhibition effects of OTC on the metagenomic-based analysis, especially nonculturable bacteria, and its effects on the biological functions of the corresponding bacteria are not well known.
In this study, we isolated and identified several common bacteria that cause endometritis.Overall, our analysis showed that the microbial communities in cows with endometritis are similarly dynamic within either animal groups (Figs. 3, 4, Supplementary Table 4 and 5) as reported by other research 25 .It is noteworthy that although www.nature.com/scientificreports/OTC stimulated the elimination of Bacteroidetes, Fusobacteria, etc., it did not eliminate all bacterial species.On the contrary, some bacteria species increased after OTC administration.The detection rates of bacteria such as Chryseobacterium indologenes, Bacillus cereus, and Corynebacterium were even higher in the NC group than in the BT group.Although some bacteria (e.g.Escherichia coli, Staphylococcus aureus, Staphylococcus haemolyticus, and Streptococcus), which was considered as pathogenic bacteria, has high detection rate or proportion, the individuals have not shown disease characteristics or have been cured.This indicates that identification by traditional bacterial culture methods has limitations for endometritis infection-associated strain identification.
In addition, simply analyzing the etiology or treatment of endometritis based on a specific pathogenic bacterial population may also be inaccurate.At the phylum level, changes in the bacterial communities between the NT group and the BT group indicated that abundances of Fusobacteria, Firmicutes, Bacteroidetes, Spirochaetae, Proteobacteria, Saccharibacteria, Chloroflexi, Verrucomicrobia, and Lentisphaerae changed the most from mild symptoms to severe illness.The changes in bacterial communities between the BT and AT groups indicated that OTC could significantly inhibit Bacteroidetes and Fusobacteria.The changes in bacterial communities between the NC and NT groups indicated that endometritis begins with changes in the abundance of Spirochaetae, Chloroflexi, and Cyanobacteria.These results seem to be contradictory.For example, Streptococcus and Staphylococcus belong to the Firmicutes phylum; Escherichia coli belongs to the Proteobacteria phylum.The abundance of Firmicutes was increased, while Proteobacteria did not change significantly after OTC treatment.This result suggests that exploring the bacteria that might lead to endometritis at the phylum level as a guideline to prevent or cure endometritis is ambiguous.However, since the database is not sufficiently comprehensive, we selected the genus level to further annotate the differences between different groups of bacteria and analyze their biological effects.
At the genus level, Sulfuricurvum, Caproiciproducens, and Lactobacillus were not detected in the uterus of healthy cattle but were in cows in the onset stage of endometritis.The abundances of bacteria such as Tepidimonas, Ornithinicoccus, and Paludibacter increased significantly during the deterioration of endometritis.Interestingly, Amphibacillus and Paraliobacillus could not be detected in the uteruses with endometritis.Amphibacillus was detectable in cows with endometritis until they were cured.Faecalibacterium, hoa5-07d05_gut_group, Salinicoccus, Methylophilus, Proteiniclasticum, Cellulomonas, and Phenylobacterium were undetectable when cows were severely ill, indicating that these bacteria could not survive in a severe endometritis environment.
In addition, we also found that no flora disappeared after OTC treatment, indicating that OTC could not completely remove existing bacteria.However, after treatment with OTC, the relative abundances of Bacteroides, Trueperella, Peptoniphilus, Parvimonas, Porphyromonas, and Fusobacterium decreased to 15.5824%, 1.0783%, 0.4276%, 0.0979%, and 0.0646%, respectively, indicating that OTC has an active inhibitory effect against these bacteria.In contrast, the relative abundance of Marinospirillum, Erysipelothrix, and Enteractinococcus increased tens of thousands of times after OTC treatment, indicating that OTC had no significant inhibitory effect on these bacteria.
Finally, we analyzed the possible biological pathways that OTC affected and its potential effects on the physiological role of uterine bacterial communities.Postpartum insufficient uterine involution, inflammatory secretions in the uterine cavity and poor drainage are important hidden dangers of endometritis.Bacteria lead to endometritis through biological pathways such as the excretory system, endocrine system, signal transduction,

Experimental animals
On a dairy farm with 442 cows, a total of 16 Holstein cows with same months of age and parity order were detected.According to the degree of illness, among all the samples, 5 cows were healthy cows (NC group), and 5 cows had mild disease (NT group).Six cows were severely ill (BT group) and became healthy after oxytetracycline hydrochloride (OTC; Aladdin, Shanghai, China) treatment (AT group).

Uterine contents collection
Before sample collection, the anus and vulva area were wiped and disinfected with a 5% potassium permanganate solution, and then the vulvae were disinfected with cotton swabs moistened with 70% alcohol.Endometrial swabs were used to collect the uterine contents as described with some modification 25,37 .In general, an improved ethylene oxide-sterilized human uterine sampling brush was placed into an infusion cannula, and advanced into the uterus and gently rotated 38 .After removal, the uterine sampling swabs was rinsed with sterile saline, and the solution samples were collected in two sterile tubes.Bacteria in one tube were stored immediately at − 80 °C until DNA extraction.Bacteria in another tube were prepared for pathogen identification.

Diagnosis of endometritis
Holstein cows with endometritis were identified based the visual observation and ultrasound testing as described with some modification.In general, cows with a foul-smelling and slightly turbid discharged vaginal mucus were diagnosed with mild disease.Cows with fetid and white/yellow vaginal purulent secretions were diagnosed as seriously diseased.Through rectal palpation, the volume of seriously diseased cow's uterine horn was increased and often accompanied by a certain degree of edema, the elasticity of the cervix and uterus deteriorated.Through ultrasound examination, cows with thickened uterine wall and irregular inner edge as well as an obvious fluid sonolucent area in the uterus were identified as diseased cow while healthy or cured cows had clear black background without significant hyperechoic areas (Fig. 1).

Isolation and biochemical identification of endometritis pathogens
Broth medium, MacConkey agar medium, blood agar base medium, and related media were obtained from Qingdao Hope Bio-Technology (Qingdao, China).Bacteria in uterine contents were cultured with the corresponding media at 37 °C incubator for 24 h for Gram staining and microscopic examination.The morphology, staining characteristics and growth characteristics of the colonies were observed and classified.Each sample was subjected to 5 biological replicate tests, and any positive results indicated that the corresponding bacteria existed in the uterine contents.The isolated bacteria obtained were identified based on morphology, Gram staining reactions and different biochemical tests using Bergey's manual for determinative bacteriology 39 .

OTC treatment and efficacy evaluation
Briefly, uterine perfusion was performed on a diseased cow with a 20% concentration of OTC (200 g of OTC in 1 L of sterile saline).The vulva was cleaned and disinfected, and then 20 mL of OTC was pushed into the uterus every 2 days by a syringe through an infusion cannula.Four episodes of perfusion was considered a treatment course.One week after stopping the treatment, a rectal examination and an ultrasound examination were performed to verify the curative effect.When a white/yellow stench of vaginal purulent secretions was still found from the orificium vaginae and an inflammatory area was detected by ultrasound examination, the treatment was ineffectual (Fig. 1I).When there was no congestion in the vaginal mucosa, the uterine mucus was clear, and there were abnormal findings on ultrasound, the cows were considered cured (Fig. 1J).

Figure 1 .
Figure 1.Observation, ultrasound, and microscopic imaging of endometritis and bacterial mixed infection of representative examples.Mildly diseased cows had red and swollen vulvae (A), and the vaginal discharge was slightly turbid and could not be drawn into filamentous mucus (B).A large amount of white (C) or yellow (D) purulent secretion with severe malodorous smell could be observed in the vaginal opening of severely diseased cows (E-H).The uterus of diseased cows showed abnormal ultrasound imaging with hyperechoic areas (I), while healthy or cured cows had clear black background (J).(K) Mixed infection with Escherichia and Streptococcus (magnification = 100×).(L) Mixed infection with Escherichia and Staphylococcus (magnification = 100×).

Figure 2 .
Figure 2. Venn diagram and abundances of the top 30 genera in the four groups.Venn diagrams showing the unique and shared genera of the bacterial communities across different groups at the genus (A) and species (B) levels.(C) Relative abundances of the top 30 bacterial genera in the four groups.Each color represents a genus, and the length of the patch represents the relative abundance ratio of all the bacterial communities in each sample.The other genera are merged in an "others" category.The category "unknown" represents a species that was not annotated with taxonomy.

Figure 3 .
Figure 3. Variations in alpha-diversity and NMDS analysis among the four groups.(A) Comparisons of the bacterial community Chao1 indexes within the different groups.(B) Comparisons of the bacterial community Shannon indexes within the different groups.*, P < 0.05; **, P < 0.01 (with Student's t-test).(C) The dots in the figure represent the samples, and the different colors represent the different groups to which they belong.The distance between points indicates the degree of difference.Closer samples on the graph have greater similarity of diversity. 5 . The relative abundances of Clostridium_sensu_stricto_6 and Tissierella were much higher (25.839 and 10.665 times, respectively) in the NC group than in the NT group, while those of Tepidimonas, Ornithinicoccus, Paludibacter, Serratia, and Rhizobium were much lower (0.016, 0.018, 0.023, 0.042, and 0.043 times, respectively).Sulfuricurvum, Caproiciproducens, and Lactobacillus could not be detected in the NC group, while Paraliobacillus and Amphibacillus were undetectable in the NT group.The abundances of Marinospirillum, Erysipelothrix, Enteractinococcus, Erysipelotrichaceae_UCG-009, and Papillibacter were much lower in the BT group than in the AT group (0.002, 0.005, 0.008, 0.010, and 0.012 times, respectively), while those of Fusobacterium, Porphyromonas, Parvimonas, Peptoniphilus, and Trueperella were much higher (1,548.265,

Figure 4 .
Figure 4. Bacterial beta diversity based on UPGMA clustering and abundances of the top 10 genera.To optimize the view, the histogram shows only the top 10 most abundant genera; the other genera are merged in an "others" category."unknown" represents a species that are not annotated with taxonomy.Closer samples with shorter branch lengths have more similar genus-level compositions.Each color represents a genus, and the length of the patch represents the relative abundance ratio of all the bacterial communities in each sample.

Figure 5 .
Figure 5. Significant changes of bacterial communities in the four groups as determined by LEfSe analysis.Taxonomic representation of statistically consistent differences within the four groups.The taxa with significantly different abundances among groups are represented by colored dots.Colored areas (NC, NT, BT, and AT) mark the most prominent bacteria found in this study.The cladogram was generated by LEfSe and indicates differences at the phylum, class, family, order, genus, and species levels in the four groups.Each successive circle represents a phylogenetic level.Only taxa meeting a linear discriminant analysis (LDA) significance threshold of > 3.5 are shown.

Figure 6 .
Figure 6.Abundances of bacterial communities changes at phylum level and related metabolic pathways with OTC treatment.(A-C) Relative abundances of bacterial phyla in four groups.Each color represents a phylum.No significantly changed phyla are merged in an "Others with N.S. " category.(D) KEGG pathways associated with significantly different abundances in the bacterial metagenomics profiles of BT and AT groups.The bars represent the proportion of each category in the data.Category differences with a P < 0.05 were considered to be significant.

Table 1 .
Pathogen identification in uterus.a-d NC = healthy cows (NC group), NT = mild diseased cows, BT = severely ill cows, AT = cows in BT group after OTC treatment.

Table 2 ,
Supplementary Table 4, and Supplementary Table

Table 2 .
Variations of dominant bacteria composition." + " represents bacteria can be detected while "−" represents bacteria is undetectable.BT = severely ill cows, AT = cows in BT group after OTC treatment.